1. Field of the Invention
In arriving at the gist underlying the concept of the instant invention, it was conceived that thiophosphoryl triamide and its linear polymers, though not members of the phosphoramide class of compounds discussed above and shown by previous workers (TVA unpublished data; German Pat. No. 142,714, July 9, 1980; German Offensive No. 2,504,193, Sept. 4, 1975) to be ineffective as a urease inhibitor, should be reinvestigated as urease activity inhibitors. I unexpectedly found that thiophosphoryl triamide and its linear polymers are excellent urease inhibitors; in fact, the thiophosphoryl triamide can be shown to be superior to phenylphosphoryl triamide, the most potent urease inhibitor known to date.
2. Description of the Prior Art
Some phosphorus, oxygen, and nitrogen-containing heterocyclic compounds of the general structure R.sub.x PO(NH.sub.2).sub.3-x, where R=NH.sub.2, OHOC.sub.6 H.sub.5, etc., and similar in structure to the thiophosphoryl amides, have been reported to be extremely effective urease inhibitors. Several researchers in the art have demonstrated that phenyl phosphorodiamidate, (C.sub.6 H.sub.5 O)PO(NH.sub.2).sub.2, is an extremely potent inhibitor of urease activity [P. Held, S. Lang, E. Tradler, M. Klepel, D. Drohne, H. J. Hartbrich, G. Rothe, H. Scheler, S. Grundmeier, and A. Trautmann, East German Pat. No. 122,177 (Cl. C05G3/08, Sept. 20, 1976), Chem. Abstracts 87:67315W; D. A. Martins and J. M. Bremner, Soil Sci. Soc. Am. J. 48, 302-305 (1984)]. As was mentioned earlier, Bayless and Millner (U.S. Pat. No. 4,242,325, 1980, and U.S. Pat. No. 4,182,881, 1980) showed that phosphoryl triamide, PO(NH.sub.2).sub.3, and a series of N-(diaminophosphinyl) arylcarboxamides also are powerful urease inhibitors. Other investigators have shown that diamidophosphoric acid, PO(NH.sub.2).sub.2 OH, and monoamidophosphoric acid, PO(NH.sub.2)(OH).sub.2, also are effective urease inhibitors [A. Barth, W. Rollka, and H. J. Michel, Wissenschaftliche Beitraege-Martin Luther Universitaet Halle Wittenberg, No. 2, pp 5-10 (1980); N. E. Dixon, C. Gazzola, J. J. Waters, R. L. Blakeley, and B. Zerner, J. Am. Chem. Soc. 97, 4131 (1975)].
Also, since the prior art teaches that phenylphosphoryl triamide and phosphoryl triamide are both effective urease inhibitors, one expects that thiophosphoryl triamide would similarly be less effective as an inhibitor since phenylthiophosphenyl diamidate is less effective as an inhibitor.
It may be possible that the two inhibitor classes have substantially the same mechanism of inhibition, to wit, reacting with the essential sulfhydryl group(s) on the active site(s) of the urease. At this time, however, I can only speculate that the inhibitory properties of the thiophosphoryl triamide and its linear polymers result either from some yet unidentified chemical properties and/or characteristics of the compounds themselves. If the mechanism is related to reacting with, or inhibiting of such sulfhydryl group(s), it might be classified as irreversible inhibition, but more probably as competitive inhibition.
Several hundred scientific papers have been published on urease since Sumner (1926) first produced the classical octahedral crystals and showed that the enzyme was a protein, but it was in 1969 that Zerner's group [R. L. Blakeley, E. C. Webb, and B. Zerner, Biochemistry 8, 1984-1990 (1969)] prepared a highly purified urease with a full specific activity and in at least a 99% homogeneous state. They established with this preparation a reproducible molecular weight (about 590,000) and proposed that the molecule contained six subunits with asparagine as the N-terminal amino acid. Although previous work [J. F. Ambrose, G. B. Kistiakowsky, and A. G. Kridl, J. Am. Chem. Soc. 73, 1232 (1951)] had indicated that four or eight essential SH-groups were involved in the urea-hydrolysis reaction, Zerner's group could only confirm that the active site SH-groups "react slowly with N-ethylmaleimide," but they were unable to define unequivocally the number of "essential SH groups" in the 590,000 molecular weight species. In addition, Kobashi et al. [K. Kobashi, J. Hase, and T. Komai, Biochem. Biophys. Res. Commun. 23, 34 (1966)], on the basis of inhibition by hydroxamic acids, suggested that the number of active sites in the 590,000 molecular weight species of sword bean urease was two. These results seem to be confirmed by the discovery that highly purified urease from jack bean [N. E. Dixon, C. Gazzola, R. L. Blakeley, and B. Zerner, J. Am. Chem. Soc. 97 4131 (1975)] and from tobacco, rice, and soybean [J. C. Polacco, Plant Science Letters 10 249-255 (1977)] contained stoichiometric amounts of nickel (two atoms per active site), demonstrating simultaneously the first biological role definitely assigned to nickel. Over the last few years considerable effort has been made to elucidate the mechanism of the urease reaction. Although attempts to demonstrate the formation of a carbamoyl-enzyme intermediate, which was postulated many years ago, have so far failed, Zerner's group [N. E. Dixon, P. W. Riddles, C. Gazzola, R. L. Blakeley, and B. Zerner, Can. J. Biochem. 58, 1335-1344 (1980)] proposed a mechanism of reaction on the basis of a carbamoyl-transfer reaction and where the substrate is activated toward nucleophilic attack by O-coordination to a Ni.sup.2+ ion. Both Ni.sup.2+ ions are involved in this proposed mechanism. A second mechanism of reaction based on the determination of kinetic isotope effects [R. Medina, T. Olleros, and H. L. Schmidt, Proc. 4th Int. Conference on Stable Isotopes, H. L. Schmidt, H. Forstel, and K. Heizinger, eds., Julich, March 1981, Elsevier, Amsterdam (1982), pp 77-82] was proposed. These results indicated the existence of an enzyme-bound carbamate intermediate and demonstrated that the enzyme-Ni-substrate complex decomposes, releasing the first NH.sub.3 in a slow, rate-limiting step.
An additional complication develops from the tendency of the urease to form polymers and isozymes changing the properties of the original monomeric enzyme and probably the mechanism of reaction [W. N. Fishbein and K. Nagarajan, Arch. Biochem. Biophys. 144, 700-714 (1971)]. Finally, the properties of soil urease differ significantly from those of ureases from other sources [J. M. Bremner and R. L. Mulvaney, Soil Enzymes, R. G. Burns, ed., Academic Press (1978), pp 149-196)]; and it is much more difficult to obtain reliable kinetic data for enzymes in heterogeneous environments, such as soil, than for enzymes in homogeneous solutions.
While many urease inhibitors have been identified, few kinetic descriptions include the type of inhibition. The reversible and competitive inhibition of sword bean urease by a wide variety of hydroxamic acids was discovered by Kobashi et al. [K. Kobashi, J. Hase, and K. Uehara, Biochim. Biophys. Acta 65, 380-383 (1962)]. Kinetic and spectral studies performed by B. Zerner and coworkers [N. E. Dixon, J. A. Hinds, A. K. Fihelly, C. Gazzola, D. J. Winzor, R. L. Blakeley, and B. Zerner, Can J. Biochem. 58, 1323-1334 (1980)] established that hydroxamic acids were reversibly bound to active-site nickel ions in jack bean urease. Chemical and physical studies of the enzymatically inactive phosphoramidate-urease complex provide convincing evidence that phosphoramidate binds reversibly to the active-site nickel ion [N. E. Dixon, R. L. Blakeley, and B. Zerner, Can. J. Biochem. 58, 481-488 (1980)].
The kinetics of urease inhibition by phenyl phosphorodiamidate (which demonstrates a competitive inhibition) and hydroquinone (which exemplifies a mixed inhibition mechanism) were performed by L. J. Youngdahl and E. R. Austin at the International Fertilizer Development Center (IFDC, unpublished results). A kinetic study of the soil urease inhibition by six substituted ureas, compounds which are used as herbicides, showed that all six compounds exhibited mixed inhibition characteristics (competitive and noncompetitive) [S. Cervelli, P. Nannipieri, G. Giovannini, and A. Perna, Pesticide Biochem. Physiol. 5, 221-225 (1975)].
There are very few additional publications on kinetic studies concerning soil ureases [J. M. Bremner and R. L. Mulvaney, Soil Enzymes, R. G. Burns, ed., Academic Press (1978), pp 149-196]. The main work in this area has been to establish the inhibitory properties of potential test compounds, irrespective of the kind of inhibition that is responsible for the retardation of the urea hydrolysis. However, the successful use of this technology by the fertilizer industry does not require that the mechanism be indentified.
Taking into consideration all of this information, one can establish that even though urease has been extensively studied for about 60 years, the mechanism of action and the mechanism of inhibition of this enzyme, especially in heterogeneous environments such as soils, are at best only partially known.